Various methods have been derived for monitoring low molecular weight compounds in blood plasma or other biological media. For example, enzyme electrodes have been used for the direct measurement of biomolecules in physiological samples, such as glucose, urea, amino acids, and others. These enzyme electrodes include a selective enzyme layer immobilized at the surface of a potentiometer or amperometeric device that senses the steady state concentration of a product formed in the immobilized layer as the substrate for the enzyme diffuses into this reactive film.
The U.S. Pat. No. 4,344,438 to Schultz, issued Aug. 17, 1982 discloses an optical sensor for monitoring low molecular weight compounds in blood plasma. The device utilizes a binding protein immobilized inside a membrane. A conjugate between glucose and fluorescein serves to generate a signal. The conjugate binds to an immobilized lectin in the absence of glucose such that there is no fluorescence in an adjacent dialysis chamber. If glucose enters into the lumen through the membrane, it competes with binding sites on the lectin and sets free glucose fluorescein conjugate, hence there is fluorescence in the detector tube. It is disadvantageous to use enzymes as signal generators in this system because the dissociated conjugate in the presence of glucose in the middle of the detector tube could provide a high background signal so that the reduced amount of conjugate on the side of the tube might not be distinguishable from the background or from the previous signal where no external glucose was present.
The U.S. Pat. No. 4,517,288 to Giegel et al, issued May 14, 1985 discloses a method for conducting a ligand assay in an inert porous medium wherein a binding material is immobilized within the medium. The method includes the steps of immobilizing a binding material within a finite zone of a medium and applying an analyte to the zone containing the immobilized binding material. A labeled indicator is applied to the zone and becomes bound within the zone in an amount which can be correlated to the amount of the analyte in the zone. A solvent is applied to the center of the zone to chromatographically separate the unbound labeled indicator from the zone and measuring the amount of labeled indicator remaining in the zone. This chromatographic method cannot be used in a continuous assay device for continually determining changes in a dynamic system.
Anderson et al reported in Clin. Chem. 34/7, 1417-1421 (1988) of a fiberoptic chemical sensor for continuous, reversible measurement of phenytoin. Beta-phycoerythrin-phenytoin and Texas Red-labeled antibody to phenytoin were sealed inside a short length of cellulose dialysis tubing. The tubing was cemented to the distal end of an optical fiber. When the sensor was alternately placed into solutions with various concentrations of free phenytoin, the drug crossed the dialysis membrane and displaced a fraction of the beta-phycor eythrin-phenytoin from the antibody. The resulting change in fluorescent signal was measured with a fiberoptic fluorometer. A reversible sensor could be made that has a response time suitable for continuous measurements utilizing the invention. In an abstract presented at the proceedings of the symposium on Sensor Science and Technology, Apr. 6-8, 1987 and published in the same proceeding abstracts by the Electrochemical Society, Inc. of Pennington, N.J., Vol. 87-15, W. Schramm, et al (the inventor of the present application) postulated an immunoglobulin-based biosensor including heterobifunctional structures that bind reversibly to immobilized counterparts. A combination of two antibody-antigen reactions hypothetically would generate a signal for continuous monitoring of analytes. The abstract hypothesizes several biosensor systems, the abstract being published prior to any attempt at reduction to practice of the concept.
The present invention provides means for constructing either a discontinuous single use disposable analytical test device or a reversible biosensor for continuous measurements of analytes. The invention utilizes the concept of a dislocated conjugate, such as the general concept being disclosed in the Shultz patent, but goes further so as to bind the conjugate at another site and measure it at that second site. Accordingly, the present invention can utilize enzymes as a signal generator. Further, the present invention can generate two signals, one at each of the two binding sites.